George Davis

IDENTIFICATION OF A NEW ANTIFUNGAL TARGET SITE THROUGH A DUAL BIOCHEMICAL AND MOLECULAR-GENETICS APPROACH

Abstract The target site of the antifungal compound LY214352 [8-chloro-4-(2-chloro-4-fluorophenoxy) quinoline] has been identified through a dual biochemical and molecular-genetics approach. In the molecular-genetics approach, a cosmid library was prepared from an Aspergillus nidulans mutant that was resistant to LY214352 because of a dominant mutation in a single gene. A single cosmid (6A6-6) that could transform an LY214352-sensitive strain of A. nidulans to LY214352-resistance was isolated from the library by sib-selection. Restriction fragments from cosmid 6A6-6 containing the functional resistance gene were identified by transformation, and sequenced. The LY214352-resistance gene coded for a protein of 520 amino acids that had a 34% identity and a 57% similarity in a 333 amino-acid overlap to E. coli dihydroorotate dehydrogenase (DHO-DH). The results of a series of biochemical mechanism-of-action studies initiated simultaneously with molecular-genetic experiments also suggested that DHO-DH was the target of LY214352. Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I50=40 ng/ml) and a highly resistant mutant (I50>100 μg/ml) conclusively demonstrated that DHO-DH is the target site of LY214352 in A. nidulans. Several mutations in the DHO-DH (pyrE) gene that resulted in resistance to LY214352 were identified.

Isolation, characterization, and genetic analysis ofAspergillus nidulans mutants resistant to the antifungal compound LY214352

We have isolated and characterized six chemically induced mutants of the filamentous fungusAspergillus nidulans that are resistant to the experimental fungicide 8-chloro-4-(2-chloro-4-fluoro-phenoxy)quinoline (LY214352). The mutants are 13- to 430-fold more resistant to LY214352 than the parental strain, and one of the mutant strains requires LY214352 for maximal growth. The resistance trait is governed by a single dominant or partially dominant gene in each mutant, and it is likely that all of the mutations are allelic. The LY214352-resistant mutants were not cross-resistant to other compounds that inhibit the growth ofA. nidulans. The implications of these findings on the potential for development of resistance to LY214352 are discussed.

Phoslactomycins from Streptomyces sp. MLA1839 and Their Biological Activities

Phoslactomycins H (1) and I (2), two new members of the phoslactomycin class of chemistry, were isolated from Streptomyces sp. MLA1839 on the basis of their antifungal activities. Their structures were elucidated using extensive NMR spectroscopy and mass spectrometry. Phoslactomycin H (1) featured a rare and unprecedented N,N-dimethylamine substitution at C-4 and existed as a hydroxy acid rather than the more common lactone. Herein, we report the structure of these compounds and their biological activities.

Alveolarides: Antifungal Peptides from Microascus alveolaris Active against Phytopathogenic Fungi

Three novel cyclodepsipeptides, alveolarides A (1), B (2), and C (3), each possessing the rare 2,3-dihydroxy-4-methyltetradecanoic acid unit and a β-phenylalanine amino acid residue, along with the known peptide scopularide were isolated and identified from the culture broth of Microascus alveolaris strain PF1466. The pure compounds were evaluated for biological activity, and alveolaride A (1) provided strong in vitro activity against the plant pathogens Pyricularia oryzae, Zymoseptoria tritici, and Ustilago maydis. Moderate activity of alveolaride A was observed under in planta conditions against Z. tritici, Puccinia triticina, and Phakopsora pachyrhizi. Structures of 1, 2, and 3 were determined by detailed analysis of NMR (1D and 2D) and mass spectrometry data. The partial absolute configuration of alveolaride A (1) was established.